To the Editor:
Gout is a common disease that is caused by urate crystals (monosodium urate monohydrate) that are formed in the body. The clinical features of gout include crystal-induced arthritis, accumulation of urate crystals to form tophi, urolithiasis, and kidney disease.
Urate crystals may be formed within the body when the concentration of urate exceeds the limit of solubility (∼7 mg/dl in plasma at 37°C). Most of the mammalian species possess the urate-degrading enzyme urate oxidase and have rather low concentrations of plasma urate. The human genome contains the sequence for urate oxidase, but the gene has lost its function because of at least 3 deleterious mutations (1, 2). Because of the deficiency of urate oxidase in the species, humans tend to show plasma urate levels that are over the limit of solubility, and some individuals with serum urate concentrations >7 mg/dl develop gout. Although hyperuricemia does not necessarily mean that gout is also present, the higher the serum urate concentration, the higher the incidence of gout. Therefore, hyperuricemia is the most important risk factor for gout.
Urate oxidase deficiency, however, is insufficient to maintain blood urate at higher levels in humans than in other mammals because of free filtration of urate at the glomerulus. Urate transporter 1 (URAT1), which is encoded by SLC22A12, has been identified as a urate-anion exchanger in humans (3). It has been demonstrated that URAT1 plays a central role in the reabsorption of urate from the glomerular filtrate and may be the major mechanism for regulating blood urate levels (3, 4).
In this study, we examined SLC22A12 in patients with gout. The aim was to analyze the roles of SLC22A12 in the development of gout.
A total of 185 male Japanese patients with primary gout (mean ± SD age 39.8 ± 11.5 years) who attended the outpatient clinic at the Institute of Rheumatology, Tokyo Women's Medical University, were randomly selected for the study. The diagnosis of acute gouty arthritis was based on the preliminary classification criteria described by Wallace et al (5). Healthy Japanese subjects who had registered for our previous study (6) and had given their consent to use their DNA samples in another study served as controls. The control group consisted of 594 men (mean ± SD age 40.7 ± 11.5 years) and 386 women (mean ± SD age 39.7 ± 11.5 years). The study was authorized by the Hospital Ethics Committee for Human Genome Research.
Serum uric acid levels were measured with a Hitachi H7700 autoanalyzer (Hitachi, Tokyo, Japan), using a colorimetric method that is based on the uricase–peroxidase method. Genomic DNA was prepared from peripheral blood. The study was focused on the G774A mutation in SLC22A12. This mutation is present in exon 4 and leads to the substitution of a stop codon for tryptophan (W258X) (3). The homozygous G774A mutation is the predominant cause of idiopathic renal hypouricemia in Japanese patients (7, 8).
The genotypes of G774A were determined using the TaqMan Assay-By-Design method (Applied Biosystems, Foster City, CA). The primer sequences were 5′-AGTGGCCTACGGTGTGC-3′ (forward) and 5′-CAGGAGTACAAAAAGCAGAGGAAGA-3′ (reverse). The sequences of the TaqMan minor groove binding (MGB) probe were 5′-FAM-CAGTGTCCAGTCCC-MGB-3′, and 5′-VIC-CAGTGTTCAGTCCC-MGB-3′.
To confirm the genotype obtained by the TaqMan assay for G774A, a polymerase chain reaction–restriction fragment length polymorphism was performed on representative samples. Ten nanograms of genomic DNA was amplified with the forward primer 5′-CCGCCTCAGCTCAGCGGGCAAGCAT-3′ and the reverse primer 5′-CCCCCGGGTGGAGAGTGGGCAGGAT-3′ (8). The genotypes were identified by Bsr I restriction endonuclease digestion (New England Biolabs, Beverly, MA), which recognizes its target sequences only when the normal allele is present.
Results are expressed as the mean ± SD. Statistical analyses were performed with Fisher's exact probability test and the Mann-Whitney U test. P values less than 0.05 were considered significant.
Table 1 shows the G774A genotypes and allele frequencies in the gout patients and healthy controls. A homozygous mutation of G774A was found in neither of the study groups. In the control group, 25 men and 20 women were heterozygous for G774A. However, none of the gout patients had the mutant allele. Consistent with the findings of a previous study (9), the frequency of the G774A mutant allele in the healthy control subjects was high (2.3%).
|Gout patients (n = 185)||Male controls (n = 594)||All controls (n = 980)||P, OR (95% CI)|
|Gout patients vs. male controls||Gout patients vs. all controls|
|G/G||185 (100.0)||569 (95.8)||935 (95.4)||0.001, 0.000||<0.001, 0.000|
|G/A||0 (0.0)||25 (4.2)||45 (4.6)||(0.000–0.496)†||(0.000–0.437)†|
|A/A||0 (0.0)||0 (0.0)||0 (0.0)|
|G||370 (100.0)||1,163 (97.9)||1,915 (97.7)||0.002, 0.000||0.025, 0.000|
|A||0 (0.0)||25 (2.1)||45 (2.3)||(0.000–0.503)||(0.000–0.828)|
When the proportions of the genotypes in the gout patients and healthy male control subjects were compared, heterozygotes (G/A) were observed more frequently in the male control subjects than in the gout patients (for G/A versus G/G, odds ratio [OR] 0.000 [95% confidence interval (95% CI) 0.000–0.496]; P = 0.001) (Table 1). When the allele frequencies were compared in these 2 groups, the gout patients had a significantly lower A allele frequency than did the healthy male control subjects (for the A allele versus the G allele, OR 0.000 [95% CI 0.000–0.503]; P = 0.002) (Table 1).
We also compared the serum levels of uric acid between controls with the G/A and G/G genotypes. There were 25 male and 20 female control subjects with the G/A genotype. Serum levels of uric acid were significantly lower in subjects with the G/A genotype than in those with the G/G genotype both in the male control group (mean ± SD 3.9 ± 0.8 for G/A and 5.8 ± 1.1 for G/G; P < 0.0001) and in the female control group (2.9 ± 0.6 for G/A and 4.0 ± 0.8 for G/G; P < 0.0001). These data indicated that the G774A mutation in the SLC22A12 gene decreased uric acid levels in men and in women.
Our study has thus shown that the G774A mutation in SLC22A12 not only lowers serum uric acid levels in both men and women, but also prevents the development of gout in men. Since all study subjects were Japanese, the role of this mutation in other ethnic groups remains to be determined. In addition, all of the gout patients in this study were men. The role of the G774A mutation in women with gout should be clarified in another study. The relationship between the G774A mutation and hypouricemia has previously been reported (7), and the findings of the present study extend the clinical significance of URAT1 to gout. More comprehensive genetic studies would be needed to investigate a different mechanism of URAT1 in the development of gout.
Gout is a common disease, and germline mutations in various genes are known to be associated with gout. Thus, mutations in the genes for hypoxanthine guanine phosphoribosyltransferase, phosphoribosylpyrophosphate synthetase, glucose-6-phosphatase, and muscle phosphofructokinase are associated with a urate overproduction type of gout. Mutations in the uromodulin gene cause a urate underexcretion type of gout. Our study has shown that there are germline mutations that promote the development of gout as well as germline mutations that prevent it.
Previous studies have suggested that gout is not a pure genetic disease with a known Mendelian mode of inheritance, but rather, various genetic and environmental factors are likely to determine the susceptibility to gout. The development of gout in a given subject, therefore, is likely to be controlled by various genes, some of which promote it and others of which prevent it. Findings of the present study indicate that the G774A mutation in the SLC22A12 gene serves as a suppressing factor for the development of gout.
Supported by a grant for Research for the Future program from the Japan Society for the Promotion of Science.